U.S. patent number 11,052,172 [Application Number 16/324,637] was granted by the patent office on 2021-07-06 for hemostatic flowable.
This patent grant is currently assigned to BIOM'UP FRANCE SAS. The grantee listed for this patent is BIOM'UP. Invention is credited to Valerie Centis, Alexia De Gasperis, Doris Moura Campos, William Spotnitz.
United States Patent |
11,052,172 |
Spotnitz , et al. |
July 6, 2021 |
Hemostatic flowable
Abstract
The invention relates to a kit to prepare an hemostatic flowable
comprising: --A hemostatic powder having a composition comprising:
.smallcircle.non-cross-linked collagen of the fibrillar type
comprising a content of fibrous collagen and/or fibrillar collagen
of at least 70% by weight relative to the total weight of the
collagen; .smallcircle.at least one monosaccharide; and
.smallcircle.at least one glycosaminoglycan; --A saline solution to
be mixed with the hemostatic powder in order to form the hemostatic
flowable. The invention also relates to a method for preparing an
hemostatic flowable with such a kit, comprising the steps of: a.
Providing the hemostatic powder in a container; b. Adding a
quantity of the saline solution in the container enclosing the
hemostatic powder, closing and shaking said container in order to
promote hydration of the hemostatic powder to form the hemostatic
flowable.
Inventors: |
Spotnitz; William (Gainesville,
FL), Centis; Valerie (Lyons, FR), Moura Campos;
Doris (Bourgoin Jallieu, FR), De Gasperis; Alexia
(Saint Genis Laval, FR) |
Applicant: |
Name |
City |
State |
Country |
Type |
BIOM'UP |
Saint-Priest |
N/A |
FR |
|
|
Assignee: |
BIOM'UP FRANCE SAS
(Saint-Priest, FR)
|
Family
ID: |
1000005658784 |
Appl.
No.: |
16/324,637 |
Filed: |
August 11, 2017 |
PCT
Filed: |
August 11, 2017 |
PCT No.: |
PCT/EP2017/070428 |
371(c)(1),(2),(4) Date: |
February 11, 2019 |
PCT
Pub. No.: |
WO2018/029340 |
PCT
Pub. Date: |
February 15, 2018 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20190167840 A1 |
Jun 6, 2019 |
|
Foreign Application Priority Data
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Aug 12, 2016 [EP] |
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16184119 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L
26/0052 (20130101); A61L 26/008 (20130101); A61L
26/0066 (20130101); A61L 2300/418 (20130101); A61L
2400/06 (20130101); A61L 2300/236 (20130101); A61L
2400/04 (20130101); A61L 2300/232 (20130101); A61L
2300/252 (20130101) |
Current International
Class: |
A61L
26/00 (20060101) |
References Cited
[Referenced By]
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|
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WO |
|
WO 2012/122044 |
|
Sep 2012 |
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WO |
|
WO 2012/146655 |
|
Nov 2012 |
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WO |
|
Other References
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1502-1518. cited by applicant.
|
Primary Examiner: Soroush; Ali
Attorney, Agent or Firm: Birch, Stewart, Kolasch &
Birch, LLP
Claims
The invention claimed is:
1. A kit to prepare an hemostatic flowable comprising: a hemostatic
powder having a composition comprising: non-cross-linked collagen
of the fibrillar type comprising a content of fibrous collagen
and/or fibrillar collagen of at least 70% by weight relative to the
total weight of the collagen; at least one monosaccharide; and at
least one glycosaminoglycan; a component which makes the hemostatic
flowable, wherein said component consists of a saline solution to
be mixed with the hemostatic powder.
2. The kit of claim 1, wherein in the composition of the hemostatic
powder: the collagen is in an amount ranging from 80% to 90% by
weight relative to the total weight of the composition of the
hemostatic powder; the at least one monosaccharide is in an amount
ranging from 1% to 12.5% by weight relative to the total weight of
the composition of the hemostatic powder; and the at least one
glycosaminoglycan is in an amount ranging from 2% to 25% by weight
relative to the total weight of the composition of the hemostatic
powder.
3. The kit of claim 1, wherein in the composition of the hemostatic
powder: the collagen is in an amount ranging from 80% to 90% by
weight relative to the total weight of the composition of the
hemostatic powder; the at least one monosaccharide is in an amount
ranging from 2.5% to 7.5% by weight relative to the total weight of
the composition of the hemostatic powder; and the at least one
glycosaminoglycan is in an amount ranging from 5% to 12.5% by
weight relative to the total weight of the composition of the
hemostatic powder.
4. The kit of claim 1, wherein in the composition of the hemostatic
powder: the collagen is in an amount ranging from 84% to 88% by
weight relative to the total weight of the composition of the
hemostatic powder; the at least one monosaccharide is in an amount
ranging from 4% to 6% by weight relative to the total weight of the
composition of the hemostatic powder; and the at least one
glycosaminoglycan is in an amount ranging from 8% to 10% by weight
relative to the total weight of the composition of the hemostatic
powder.
5. The kit of claim 1, wherein in the composition of the hemostatic
powder, the at least one monosaccharide is glucose and the at least
one glycosaminoglycan is chondroitin sulfate.
6. The kit of claim 1, wherein in the composition of the hemostatic
powder, the at least one glycosaminoglycan is chosen among
chondroitin sulfate, dermatan sulfate, hyaluronic acid and mixtures
thereof.
7. The kit of claim 1, wherein the composition of the hemostatic
powder further comprises at least one coagulation factor in an
amount lower than 0.1% by weight relative to the total weight of
the composition of the hemostatic powder.
8. The kit of claim 7, wherein the coagulation factor is
thrombin.
9. The kit of claim 1, wherein the saline solution comprises
distilled water and sodium chloride, wherein the sodium chloride is
in an amount ranging from 0.5% to 1.5% by weight relative to the
total weight of the saline solution, most preferably in an amount
of 0.9% by weight relative to the total weight of the saline
solution.
10. The kit of claim 1, comprising between 1 g and 2 g of the
hemostatic powder and 4 mL and 10 mL of saline solution.
11. The kit of claim 1, wherein the mass of the saline solution is
between 2 to 10 times of the mass of the hemostatic powder, and
preferably between 4 to 5 times of the mass of the hemostatic
powder.
12. The kit of claim 1, wherein the hemostatic powder is enclosed
in a dispenser having a container portion formed with bellows and a
nozzle portion arranged on the container portion, wherein the
nozzle portion has an opening for filing the container portion with
the saline solution to be mixed with the hemostatic powder in order
to form the hemostatic flowable.
13. The kit of claim 12, wherein the container portion has a shape
designed to promote hydration of the hemostatic powder with the
saline solution in order to form the hemostatic flowable.
14. The kit of claim 12, wherein the dispenser further comprises a
cap designed to be removably coupled to the nozzle portion in order
to close any opening to/from the container portion.
15. A method for preparing an hemostatic flowable using the kit of
claim 1, comprising the steps of: a. providing the hemostatic
powder of the kit in a container; b. adding a quantity of the
saline solution to the container and closing and shaking said
container to promote hydration of the hemostatic powder to form the
hemostatic flowable.
16. The method of claim 15, further comprising a subsequent step c)
wherein the container enclosing the hemostatic flowable is left to
stand during a certain rest period.
17. The method of claim 15, wherein in step b), the quantity of
saline solution added in the container is between 50% and 100% of
the quantity of the saline solution provided in the kit for forming
an hemostatic flowable.
18. The method of claim 15, wherein in step b), the quantity of
saline solution added in the container is between 5 mL and 10
mL.
19. The method of claim 15, wherein in step b), the quantity of
saline solution added in the container is of 7 mL.
20. The method of claim 15, wherein in step b), the container is
shaken during between 10 seconds and 30 seconds.
21. The method of claim 15, wherein in step b), the container is
shaken during 20 seconds.
22. The method of claim 16, wherein in step c), the rest period is
at least of 30 seconds, preferably at least of 60 seconds, and even
more preferably at least of 90 seconds.
23. The method of claim 16, wherein in step c), the rest period is
at least of 60 seconds.
24. The method of claim 16, wherein in step c), the rest period is
at least of 90 seconds.
25. The method of claim 16, wherein in step c), the rest period is
between 30 seconds and 120 seconds.
26. The method of claim 15, wherein in step a), a quantity of 1.65
mg of hemostatic powder is provided in the container; in step b),
the quantity of saline solution added in the container is of 7 mL
and the container is shaken during 20 seconds.
27. The kit of claim 2, wherein in the composition of the
hemostatic powder, the at least one monosaccharide is glucose and
the at least one glycosaminoglycan is chondroitin sulfate.
28. The kit of claim 2, wherein the composition of the hemostatic
powder further comprises at least one coagulation factor in an
amount lower than 0.1% by weight relative to the total weight of
the composition of the hemostatic powder.
29. A kit to prepare an hemostatic flowable comprising: a
hemostatic powder having a composition comprising: collagen of the
fibrillar type comprising a content of fibrous collagen and/or
fibrillar collagen of at least 70% by weight relative to the total
weight of the collagen; at least one monosaccharide; and at least
one glycosaminoglycan; a component which makes the hemostatic
flowable, wherein said component consists of a saline solution to
be mixed with the hemostatic powder, wherein the hemostatic
flowable has a viscosity of 20 Pas to 10000 Pas when the hemostatic
powder is mixed with the saline solution.
30. A kit to prepare an hemostatic flowable comprising: a
hemostatic powder having a composition comprising: non-cross-linked
collagen of the fibrillar type comprising a content of fibrous
collagen and/or fibrillar collagen of at least 70% by weight
relative to the total weight of the collagen; at least one
monosaccharide; and at least one glycosaminoglycan; a component
which makes the hemostatic flowable, wherein said component
consists of a saline solution to be mixed with the hemostatic
powder, wherein the hemostatic flowable has a viscosity of 20 Pas
to 10000 Pas when the hemostatic powder is mixed with the saline
solution.
Description
FIELD OF THE INVENTION
The present invention relates to the field of hemostatic
compositions, to the use of specific compounds or compositions as a
hemostatic agent, to a method for preparing a hemostatic
composition and to a hemostatic method.
TECHNICAL BACKGROUND
Wounds, whether external or internal, traumatic or surgical,
frequently lead to bleeding. Such bleeding can be more or less
significant. Bleeding is prevented and stopped via a set of
physiological phenomena called "hemostasis". Hemostasis helps
repair the vascular breach and, generally, ensures the maintenance
of vessel and tissue integrity.
When a blood vessel is injured, a natural mechanism comprising
various stages is triggered to stem the flow of blood. First,
vasoconstriction, which slows the bleeding, lasts for 15 to 60
seconds and induces a complex cascade of reactions. A fibrous mesh
composed of fibrin forms around the platelet plug: the final
thrombus is formed and is protected from premature dissolution by
factor XIII, which stabilizes fibrin. Finally, the fibrin mesh
draws tighter (retraction) and the edges of the wound come
together: the wound shrinks. Within the stable, cross-linked
fibrin, fibroblasts can then grow and organize into a conjunctive
matrix within the thrombus and finally close the wound.
No solid fibrin is present in circulating blood; if it were it
would immediately obstruct vital vessels. However, fibrin's
precursor, fibrinogen, is present. Under the action of thrombin,
whose synthesis is activated by coagulation factors, fibrinogen is
transformed into insoluble fibrin.
Lastly, several days or weeks after successful healing of the
wound, the fibrin cluster is destroyed during fibrinolysis.
In spite of this biochemical phenomenon, it is often necessary, in
particular in the case of wounds that are too large or in the case
of diffuse bleeding, to "artificially" carry out hemostasis.
There are "mechanical" solutions to help obtain hemostasis, such as
pressure, ligature and electrocoagulation, which are used as
first-line treatments. However, these solutions have little or no
effectiveness in a certain number of cases, such as oozing
capillary hemorrhages, hemorrhages of hypervascularized organs such
as the spleen or liver, hemorrhages leading to diffuse bleeding,
for example bones, and/or in neurosurgery.
"Chemical" solutions, in particular implemented in certain current
hemostatic products, also exist. The components of said chemical
solutions are in general either of the "absorbent" or "active"
type.
Absorbent hemostatic products, notably comprising polysaccharides
such as regenerated oxidized cellulose or alginates, function
mainly by mechanical action and simple absorption. They frequently
present a problem of excessive swelling. If said swelling leads to
rapid absorption of liquid, in particular blood, it can also lead
to undesirable pressure when used in a "closed" environment, for
example in contact with the dura mater or in urology.
In addition, certain products, notably those comprising plant
polysaccharides such as cellulose or alginates, can further cause
inflammatory reactions during their resorption and/or can lead to
degradation products not recognized by the host. The consequence of
this is that it is desirable to remove such products so that they
do not remain in the body and thus do not produce these adverse
effects.
Active hemostatic products, such as products containing thrombin or
fibrin, are often blood-derived products. Such products involve
risks of allergies and disease transmission, in particular in the
case where the disease vector would not be inactivated by
classically applied treatments. In addition, said downstream
treatments are generally complex and/or costly. Lastly, in general
they can require preparation before use, which can be a constraint,
indeed a nuisance, in terms of an emergency.
Moreover, products containing both fibrin and thrombin base their
mode of action on the interaction between the two blood-derived
products comprising the product. The reaction can occasionally take
place without interaction with the blood, in which case the
products are said to float. In other words, the product is pushed
away by the blood which continues to flow, possibly causing the
product to become diluted or to coagulate and form a gel on top of
the blood, a situation in which the flow of blood is not blocked.
Hemostasis can thus not be achieved.
An hemostatic powder, its method of production and method of use,
have been disclosed in the international application published
under the reference WO 2012/146655 on 1 Nov. 2012, the content of
which is entirely incorporated by reference in the present
application.
Such hemostatic powder has a satisfactory absorption capacity, good
hemostatic capacity, almost no adverse effects, good capacity to
anchor on the edge of the wound and satisfactory penetration in the
blood flow where it is used and/or limited swelling.
In addition to these good hemostatic properties, such hemostatic
powder presents the advantage of having a very good flowability
that enables it to be sprayed on the bleeding region. It can be
administered in most surgical procedures, such as laparotomies,
laparoscopies, coelioscopies, and robotic surgical techniques
The hemostatic powder can be directly applicable on the bleeding
region without specific preparation by the surgeon which is another
advantage.
It might sometimes be necessary to use specific powder dispensers
to ease the application of the hemostatic powder on a very specific
bleeding region.
The aim of the present invention is to propose an hemostatic
product that is simple to use, and especially does not need a
complex preparation process, which can further be easily applied on
a specific area to cover the whole bleeding region of interest.
Another aim of the present invention is to propose an hemostatic
product that has a good hemostasis efficacy, and an enhanced
efficacy compared to existing hemostatic products.
Still another aim of the present invention is to propose a kit for
preparing an hemostatic product that can be used immediately
without extensive preparation, with minimal handling of the
hemostatic product, and which can be, for example, of used in
several surgical procedures.
SUMMARY OF THE INVENTION
To this end, we propose a kit and a method for preparing an
hemostatic flowable as defined in the appended claims.
More specifically, we propose a kit to prepare an hemostatic
flowable comprising: A hemostatic powder having a composition
comprising: collagen of the fibrillar type comprising a content of
fibrous collagen and/or fibrillar collagen of at least 70% by
weight relative to the total weight of the collagen; at least one
monosaccharide; and at least one glycosaminoglycan; A saline
solution to be mixed with the hemostatic powder in order to form
the hemostatic flowable.
Preferably, the collagen used for the hemostatic powder composition
is not cross-linked. Using non-cross-linked collagen in the
composition aims in particular at simplifying the manufacturing
process.
In another preferred aspect, the saline solution is pure,
consisting in a mix of distilled water and sodium chloride only,
meaning that there is no additives component in the solution.
Preferable but non-limiting aspects of such a kit, taken alone or
in combination, are the following: in the composition of the
hemostatic powder: the collagen is in an amount ranging from 80% to
90% by weight relative to the total weight of the composition of
the hemostatic powder; the at least one monosaccharide is in an
amount ranging from 1% to 12.5% by weight relative to the total
weight of the composition of the hemostatic powder; and the at
least one glycosaminoglycan is in an amount ranging from 2% to 25%
by weight relative to the total weight of the composition of the
hemostatic powder. in the composition of the hemostatic powder: the
collagen is in an amount ranging from 80% to 90% by weight relative
to the total weight of the composition of the hemostatic powder;
the at least one monosaccharide is in an amount ranging from 2.5%
to 7.5% by weight relative to the total weight of the composition
of the hemostatic powder; and the at least one glycosaminoglycan is
in an amount ranging from 5% to 12.5% by weight relative to the
total weight of the composition of the hemostatic powder. in the
composition of the hemostatic powder: the collagen is in an amount
ranging from 84% to 88% by weight relative to the total weight of
the composition of the hemostatic powder; the at least one
monosaccharide is in an amount ranging from 4% to 6% by weight
relative to the total weight of the composition of the hemostatic
powder; and the at least one glycosaminoglycan is in an amount
ranging from 8% to 10% by weight relative to the total weight of
the composition of the hemostatic powder. in the composition of the
hemostatic powder, the at least one monosaccharide is glucose and
the at least one glycosaminoglycan is chondroitin sulfate. in the
composition of the hemostatic powder, the at least one
glycosaminoglycan is chosen among chondroitin sulfate, dermatan
sulfate, hyaluronic acid and mixtures thereof. the composition of
the hemostatic powder further comprises at least one coagulation
factor in an amount lower than 0.1% by weight relative to the total
weight of the composition of the hemostatic powder. the coagulation
factor is thrombin. the saline solution comprises--or consists
of--distilled water and sodium chloride, wherein the sodium
chloride is in an amount ranging from 0.5% to 1.5% by weight
relative to the total weight of the saline solution, most
preferably in an amount of 0.9% by weight relative to the total
weight of the saline solution. the kit comprises between 1 g and 2
g of the hemostatic powder and 4 mL and 10 mL of saline solution.
the mass of the saline solution is between 2 to 10 times of the
mass of the hemostatic powder, and preferably between 4 to 5 times
of the mass of the hemostatic powder. the hemostatic powder is
enclosed in a dispenser having a container portion formed with
bellows and a nozzle portion arranged on the container portion,
wherein the nozzle portion has an opening for filing the container
portion with the saline solution to be mixed with the hemostatic
powder in order to form the hemostatic flowable. the container
portion has a shape designed to promote hydration of the hemostatic
powder with the saline solution in order to form the hemostatic
flowable. the dispenser further comprises a cap designed to be
removably coupled to the nozzle portion in order to close any
opening to/from the container portion.
We also propose a method for preparing an hemostatic flowable with
the above kit, comprising the steps of: a. Providing the hemostatic
powder in a container; b. Adding a quantity of the saline solution
in the container enclosing the hemostatic powder, closing and
shaking said container in order to promote hydration of the
hemostatic powder to form the hemostatic flowable.
Preferable but non-limiting aspects of such a method, taken alone
or in combination, are the following: the method comprises a
subsequent step c) wherein the container enclosing the hemostatic
flowable is left to stand during a certain rest period. in step b),
the quantity of saline solution added in the container is between
50% and 100% of the quantity of the saline solution of the kit. in
step b), the quantity of saline solution added in the container is
between 5 mL and 10 mL, and is preferably of 7 mL. in step b), the
container is shaken during between 10 seconds and 30 seconds, and
preferably during 20 seconds. in step c), the rest period is at
least of 30 seconds, preferably at least of 60 seconds, and even
more preferably at least of 90 seconds. in step c), the rest period
is between 30 seconds and 120 seconds, preferably 90 seconds.
BRIEF DESCRIPTION OF THE DRAWINGS
Other characteristics and advantages of the invention will become
clear from the following description which is only given for
illustrative purposes and is in no way limitative and should be
read with reference to the attached drawings on which:
FIG. 1 is an illustration of a dispenser used to apply the
hemostatic flowable presented here;
FIG. 2 is an illustration of the dispenser of FIG. 1 with the cap
being open;
FIG. 3 is a schematic side view of the cap of the dispenser of FIG.
1;
FIG. 4 is a schematic side view of the nozzle portion of the
dispenser of FIG. 1;
FIG. 5 is a cross-section of the container portion of the dispenser
of FIG. 1;
FIG. 6 is an example of a result of an electrophoresis as described
in example 10.
DETAILED DESCRIPTION OF THE INVENTION
In the following description, absent a statement to the contrary,
weight percentages are given relative to the total dry weight of
the composition of the hemostatic powder.
In the context of the present invention, "total dry weight of the
composition of the hemostatic powder" refers to the total weight of
the composition of the hemostatic powder free of solvent, in
particular water, and thus the total weight relative to the
anhydrous product.
In addition, the weights of the components and the resulting
percentages can correspond to the anhydrous weight of these
components, in other words, to the weight of the component not
including the water which it could contain. This can also be
applied to the percentages obtained.
The composition of the hemostatic powder can comprise a collagen
content greater than or equal to 70% by weight relative to the
total weight of the composition of the hemostatic powder, in
particular greater than or equal to 75% by weight, in particular
greater than or equal to 77% by weight, indeed greater than or
equal to 80% by weight.
In addition, the composition of the hemostatic powder can comprise
a collagen content less than or equal to 99% by weight relative to
the total weight of the composition of the hemostatic powder, in
particular less than or equal to 96% by weight, in particular less
than or equal to 93% by weight, indeed less than or equal to 90% by
weight.
Thus, the composition of the hemostatic powder can comprise a
collagen content ranging from 70% to 99% by weight relative to the
total weight of the composition of the hemostatic powder, in
particular ranging from 75% to 96% by weight, in particular ranging
from 77% to 93% by weight, indeed ranging from 80% to 90% by
weight. Preferably, the content of collagen is around 86% by weight
of the total weight of the composition of the hemostatic
powder.
Collagen is the main structure protein in mammals. Collagen
consists of tropocollagen (TC) molecules that have lengths around
280-300 nm and diameters of around 1.5 nm.
The term "fibrous collagen" refers to collagen in the form of
fiber, corresponding to an assembly of fibrils. Fibers generally
have a diameter ranging from 1 .mu.m to 10 .mu.m. The term
"fibrillar collagen" refers to collagen in the form of fibrils.
More precisely, fibrils generally have a diameter of 10 nm to 1
.mu.m. Thus, fibrils are formed from staggered arrays of
tropocollagen molecules, and these fibrils may be arranged to form
collagen fibers. Fibrous and/or fibrillar collagen is generally not
soluble, whereas non-fibrillar collagen is highly soluble.
The definition of fibrous collagen and fibrillar collagen can be in
particular that given by Markus Buehler in "Nature designs tough
collagen: explaining the nanostructure of collagen fibrils," in
PNAS, Aug. 15, 2006, vol. 103, no. 33, pp. 12285-12290.
More than 28 different collagens have been discovered and are
classified in 3 main categories: collagens of the fibrillar type,
collagens of the non-fibrillar type, and FACIT collagens.
Collagens of the fibrillar type are collagens that mostly comprise
fibrillar and/or fibrous collagens and hardly any non-fibrillar
collagens (for example collagen of type I). Similarly, collagens of
the non-fibrillar type are collagens that mostly comprise
non-fibrillar collagens. Some collagens of the non-fibrillar type
may consist only in non-fibrillar collagens (for example collagen
of type IV or V).
The industrial extraction and purification of collagen generally
consists in the destructuration of the initial tissues to 1) remove
every or the majority of contaminant proteins and 2) to obtain the
requested structuration level depending on the final use of the
product. Collagen extraction is generally performed in acid or
basic conditions that allow the solubilisation of monomolecular
soluble collagen which is not fibrillar. The final collagen
naturally contains a mix of fibrillar/fibrous collagen and
non-fibrillar collagen. The proportion between fibrillar/fibrous
collagen and non-fibrillar collagen depends on the tissue chosen
for the extraction and the extraction process.
The final product is different than a collagen that has been
obtained by an artificial mix of only fibrillar collagen and only
non-fibrillar collagen. In the article entitled "Extraction of
collagen from connective tissue by neutral salt solutions"
(Proceedings of the NATIONAL ACADEMY OF SCIENCES Volume 41 Number I
Jan. 15, 1955 by Jerome Gross, John H. Highberger and Francis O.
Schmitt), are shown the differences between fibrillar and
non-fibrillar collagens obtained after a specific extraction
process which leads--as described previously--to a mix of those two
collagens.
In the present hemostatic powder, the collagen is of the fibrillar
type, and comprises fibrous and/or fibrillar collagen in an amount
of at least 60% by weight, in particular at least 70% by weight, in
particular at least 75% by weight, indeed at least 80% by weight
relative to the total weight of the collagen.
More particularly, the collagen comprises at least 85%, in
particular at least 90%, in particular at least 95%, indeed at
least 98% by weight of fibrous and/or fibrillar collagen relative
to the total weight of the collagen in the composition of the
hemostatic powder.
Preferably the composition comprises a content of fibrous and/or
fibrillar collagen ranging from 85% to 95% by weight relative to
the total weight of the collagen in the composition, and most
preferably from 85% to 90% by weight.
This means that in the preferred embodiment, the composition of the
hemostatic powder thus comprises a content of non-fibrillar
collagen ranging from 5% to 15% by weight relative to the total
weight of the collagen in the composition, and most preferably from
10% to 15% by weight.
It is very advantageous to have a composition with such proportion
of fibrous and/or fibrillar collagen relative to the non-fibrillar
collagen, in particular for use as a hemostatic powder preparation.
Indeed, the fibrous and/or fibrillar collagen should be present in
a sufficient amount to perform the hemostasis, and the
non-fibrillar collagen should also be in a sufficient amount for
the cohesion of the product and not in a too large amount to avoid
excess of swelling.
The collagen can be selected among type I collagens or type I and
III collagens. The collagen can be extracted from various source
tissues, in particular skin and/or tendons, from all species, more
particularly porcine, bovine or equine species.
The collagen can mostly be made of fibrous collagen of porcine
origin extracted from skin and/or tendons. In the case of collagen
extracted from tendons, the extraction can be such as described in
international application WO 2010/125086.
The aforesaid collagen, in particular fibrous and/or fibrillar
collagen, can come from acid or basic extraction. According to a
particular embodiment, said collagen comes from basic extraction.
According to a particular embodiment, the collagen can be such as
described in patent application FR2944706.
Preferably, the collagen comes from a basic extraction that enables
maximizing the content of fibrous and/or fibrillar collagen in the
extracted collagen. Further, such basic extraction can be optimized
for controlling the proportion of the fibrillar/fibrous collagen
and the non-fibrillar collagen within the extracted collagen.
Unlike the acidic extraction, the basic extraction allows the
hydrolysis of proteoglycans. This action leads to the
destructuration of the tissue and the separation of the fibers
without modification of their shape. In acidic conditions, the
swelling of the inner collagen molecules in the fibers leads to
their partial destructuration during the process with the release
of greater amount of non-fibrillar soluble collagen.
The collagen can be used as it is after extraction, i.e. without
further treatment, or it can be cross-linked, notably by classic
modes of cross-linking such as thermal dehydration, the use of
bridging agents, for example formaldehyde and/or glutaraldehyde; by
oxidized polysaccharides, for example according to the method
described in international application WO 2010/125086; and/or by
oxidized amylopectins or glycogen. Cross-linking the collagen is
however not preferred as it complexifies the manufacturing process,
without necessarily increasing the hemostatic efficacy.
Preferably, the collagen used in the composition does thus not
undergo any further treatment, and in particular it is not
cross-linked. Using non-cross-linked collagen has notably the
advantage of simplifying the manufacturing process.
The composition of the hemostatic powder comprises at least one
monosaccharide, alone or in mixture with other monosaccharides.
Said monosaccharides can be selected from ribose, sucrose,
fructose, glucose and mixtures thereof. The monosaccharide present
in the composition of the invention, alone or in mixture with
monosaccharides, is in particular glucose.
The composition of the hemostatic powder can comprise a
monosaccharide content ranging from 1% to 12.5% by weight relative
to the total weight of the composition, in particular ranging from
1.5% to 10% by weight, in particular ranging from 2% to 8% by
weight, and quite particularly ranging from 2.5% to 7.5% by weight.
Most preferably, the monosaccharide content is around 5% by weight
relative to the total weight of the composition.
The composition of the hemostatic powder can comprise a
collagen/monosaccharide weight ratio ranging from 5 to 100, in
particular from 7 to 65, more particularly from 10 to 50, and still
more particularly from 11 to 40. Most preferably, the composition
comprises a collagen/monosaccharide weight ratio of around 19.
The monosaccharide, notably ribose, sucrose, fructose, glucose and
mixtures thereof, and in particular glucose, can notably make it
possible to obtain particles comprising mainly fibrous and/or
fibrillar collagen and monosaccharides with the desired
characteristics, notably of size and density. Incorporation of
monosaccharide in the mixture of collagen further allows reduction
of the electrical charges within the composition, which enables
forming a powder adapted to be placed within container such as
tubes, blower, spraying or application dispensers.
Quite particularly, the presence of monosaccharide can make it
easier and/or cheaper to obtain particles of a desired density
and/or size, in particular in terms of improving the hemostatic
properties of a powder of the composition.
Grounding collagen fibers without any additives leads to the
reduction of the size of the fibers and lowers the density of the
powder. Further, the final preparation contains important amount of
electrical charges that prevent the manipulation of the final
product. Adding monosaccharide before grinding of the collagen
leads to a hardening of the preparation to mix allowing a rapid
grinding (limitation of denaturation), thus enabling preparation of
a powder with reduced electrical charges (suitable for placing the
powder into containers, such as dispensers) and a final density
suitable for applying and reconstituting the composition.
Unlike what could have been expected such adjunction of
monosaccharide has no effect on the final activity of the product.
In particular, it does not modify the bioactivity of the final
product. The monosaccharide has no hemostatic effects.
Further, such adjunction of the monosaccharide does not make it
behaving as a foaming agent as it is the case in WO 01/97873. In WO
01/97873, the heating of the diluted solution leads to the
formation of gelatin. High concentration of gelatin can be made to
obtain very concentrated solution, but the final product contains
gelatin and not collagen. Gelatin is known to be less hemostatic
than collagen as platelet aggregation needs the presence of
collagen fibrils and structure of the native collagen which are
absent in gelatin.
According to one embodiment, the composition comprises, preferably
consists of, particles comprising, preferably consisting of,
collagen and monosaccharide, notably selected from ribose, sucrose,
fructose, glucose and mixtures thereof, in particular glucose.
The composition can comprise at least one coagulation factor. Said
coagulation factors are well known to those persons skilled in the
art. Preferably, one of the coagulation factors is thrombin. Even
more preferably, the composition of the hemostatic powder comprises
only thrombin as coagulation factor.
Said coagulation factor, in particular thrombin, can come from
animal sources (extracted from animal tissues and fluids) or from
recombinant sources (produced by cultures of genetically modified
cells). The coagulation factor may for example be thrombin
extracted from human tissues and fluids.
When a coagulation factor, in particular thrombin, is present, its
content is preferably less than 0.1% by weight relative to the
total weight of the composition of the hemostatic powder.
In the case of thrombin, international units (IU) are generally
used. Thus, the composition can comprise a thrombin content ranging
from 0.01 IU/mg to 20 IU/mg of the composition, in particular from
0.05 IU/mg to 10 IU/mg, in particular from 0.1 IU/mg to 5 IU/mg,
indeed from 0.2 IU/mg to 2 IU/mg. Most preferably the content of
thrombin--if any--is around 0.83 IU/mg of the composition.
In addition to the monosaccharide, the composition can comprise at
least one other carbohydrate compound, which can be a
glycosaminoglycan. Such carbohydrate compound may be part of the
composition, with or without a coagulation factor such as
thrombin.
Said glycosaminoglycan can be selected from chondroitin sulfates,
dermatan sulfate, hyaluronic acid and mixtures thereof, in
particular chondroitin sulfates.
Glycosaminoglycans can make it possible to improve the speed at
which blood is absorbed by the hemostatic composition. More
particularly, glycosaminoglycans can accelerate contact between the
blood and the hemostatic products, in particular collagen and
thrombin.
The composition can comprise a glycosaminoglycan content ranging
from 2% to 25% by weight relative to the total weight of the
composition, in particular ranging from 3% to 20% by weight, in
particular ranging from 4% to 15% by weight, quite particularly
ranging from 5% to 12.5% by weight. Most preferably the content of
glycosaminoglycan--if any--is around 9% by weight of the total
weight of the composition.
The composition can comprise a collagen/glycosaminoglycan weight
ratio ranging from 2.5 to 50, in particular from 3.5 to 35, more
particularly from 5 to 25, and still more particularly from 6.5 to
20.
According to one embodiment, the composition comprises at least
one, in particular one, monosaccharide and at least one, in
particular one, glycosaminoglycan, notably such as defined above,
and in particular in the amounts defined above.
The carbohydrate compounds are quite particularly monosaccharides
and glycosaminoglycans.
The composition can comprise a carbohydrate content ranging from 2%
to 25% by weight relative to the total weight of the composition,
in particular ranging from 5% to 23% by weight, in particular
ranging from 7% to 21% by weight, quite particularly ranging from
10% to 18% by weight.
The composition can comprise a collagen/carbohydrate compound
weight ratio ranging from 2 to 40, in particular from 2.5 to 30,
more particularly from 3 to 20, and still more particularly from
3.5 to 15.
The expression "total weight of carbohydrate compounds" refers to
the sum of the weight of the monosaccharides defined above and the
weight of the other carbohydrate compounds mentioned above.
According to one embodiment, the composition comprises, preferably
consists of: collagen comprising mainly a fibrous and/or fibrillar
collagen content of at least 50% by weight relative to the total
weight of the collagen, and at least one, in particular one,
monosaccharide.
Quite particularly, the composition comprises, preferably consists
of: collagen, notably in an amount ranging from 70% to 99% by
weight relative to the total weight of the composition, in
particular ranging from 75% to 96% by weight, in particular ranging
from 77% to 93% by weight, indeed ranging from 80% to 90% by
weight, wherein said collagen comprises a fibrous and/or fibrillar
collagen content of at least 50% by weight relative to the total
weight of the collagen, and at least one monosaccharide, in
particular glucose, in an amount ranging from 1% to 12.5% by weight
relative to the total weight of the composition, notably ranging
from 1.5% to 10% by weight, in particular ranging from 2% to 8% by
weight, and quite particularly ranging from 2.5% to 7.5% by
weight.
According to another embodiment, the composition comprises,
preferably consists of: collagen comprising mainly a fibrous and/or
fibrillar collagen content of at least 50% by weight relative to
the total weight of the collagen, at least one, in particular one,
monosaccharide, at least one, in particular one, coagulation
factor.
Quite particularly, the composition comprises, preferably consists
of: collagen, notably in an amount ranging from 70% to 99% by
weight relative to the total weight of the composition, in
particular ranging from 75% to 96% by weight, in particular ranging
from 77% to 93% by weight, indeed ranging from 80% to 90% by
weight, wherein said collagen content comprises a fibrous and/or
fibrillar collagen content of at least 50% by weight relative to
the total weight of the collagen, at least one monosaccharide, in
particular glucose, in an amount ranging from 1% to 12.5% by weight
relative to the total weight of the composition, in particular
ranging from 1.5% to 10% by weight, in particular ranging from 2%
to 8% by weight, and quite particularly ranging from 2.5% to 7.5%
by weight, and at least one, in particular one, coagulation factor,
in particular thrombin, in an amount ranging from 0.01 IU/mg to 20
IU/mg of the composition, in particular from 0.05 IU/mg to 10
IU/mg, in particular from 0.1 IU/mg to 5 IU/mg, indeed from 0.2
IU/mg to 2 IU/mg.
According to another embodiment, the composition comprises,
preferably consists of: collagen comprising mainly a fibrous and/or
fibrillar collagen content of at least 50% by weight relative to
the total weight of the collagen, at least one, in particular one,
monosaccharide, and at least one, in particular one,
glycosaminoglycan.
Quite particularly, the composition comprises, preferably consists
of: collagen, notably in an amount ranging from 70% to 99% by
weight relative to the total weight of the composition, in
particular ranging from 75% to 96% by weight, in particular ranging
from 77% to 93% by weight, indeed ranging from 80% to 90% by
weight, wherein said collagen content comprises a fibrous and/or
fibrillar collagen content of at least 50% by weight relative to
the total weight of the collagen, at least one monosaccharide, in
particular glucose, in an amount ranging from 1% to 10% by weight
relative to the total weight of the composition, in particular
ranging from 1% to 12.5% by weight, in particular ranging from 1.5%
to 10% by weight, in particular ranging from 2% to 8% by weight,
and quite particularly ranging from 2.5% to 7.5% by weight, and at
least one glycosaminoglycan, in particular chondroitin sulfate, in
an amount ranging from 2% to 25% by weight relative to the total
weight of the composition, in particular ranging from 3% to 20% by
weight, in particular ranging from 4% to 15% by weight, quite
particularly ranging from 5% to 12.5% by weight.
According to still another embodiment, the composition comprises,
preferably consists of: collagen comprising a fibrous and/or
fibrillar collagen content of at least 50% by weight relative to
the total weight of the collagen, at least one, in particular one,
monosaccharide, at least one, in particular one, coagulation
factor, and at least one, in particular one, glycosaminoglycan.
Quite particularly, the composition comprises, preferably consists
of: collagen, notably in an amount ranging from 70% to 99% by
weight, in particular ranging from 75% to 96% by weight, in
particular ranging from 77% to 93% by weight, indeed ranging from
80% to 90% by weight relative to the total weight, in particular to
the dry weight, of the composition, wherein said collagen comprises
a fibrous and/or fibrillar collagen content of at least 50% by
weight relative to the total weight of the collagen, at least one
monosaccharide, in particular glucose, in an amount ranging from 1%
to 10% by weight relative to the total weight of the composition,
notably ranging from 1% to 12.5% by weight, notably ranging from
1.5% to 10% by weight, in particular ranging from 2% to 8% by
weight, and quite particularly ranging from 2.5% to 7.5% by weight,
at least one coagulation factor, in particular thrombin, in an
amount ranging from 0.01 IU/mg to 20 IU/mg of the composition, in
particular from 0.05 IU/mg to 10 IU/mg, in particular from 0.1
IU/mg to 5 IU/mg, indeed from 0.2 IU/mg to 2 IU/mg, and at least
one glycosaminoglycan, in particular chondroitin sulfate, in an
amount ranging from 2% to 25% by weight relative to the total
weight of the composition, notably ranging from 3% to 20% by
weight, in particular ranging from 4% to 15% by weight, quite
particularly ranging from 5% to 12.5% by weight.
According to a quite particular embodiment, the composition
comprises, preferably consists of: collagen of the fibrillar type,
mostly comprising fibrous and/or fibrillar collagen, said collagen
of the fibrillar type being for example obtained by extraction in
basic medium, and being in an amount of around 85% by weight
relative to the total weight of the composition, glucose, in an
amount of around 4.9% by weight relative to the total weight of the
composition, thrombin, in an amount of 0.2 IU/mg to 2 IU/mg of the
composition, and chondroitin sulfate, in an amount of around 10% by
weight relative to the total weight of the composition.
According to another particular embodiment, the composition
comprises, preferably consists of: collagen of the fibrillar type,
mostly comprising fibrous and/or fibrillar collagen, said collagen
of the fibrillar type being for example obtained by extraction in
basic medium, and being in an amount of around 85% by weight
relative to the total weight of the composition, glucose, in an
amount of 5% by weight relative to the total weight of the
composition, and chondroitin sulfate, in an amount of 10% by weight
relative to the total weight of the composition.
In the context of the present invention, the expression "an amount
of around X %" refers to a variation of plus or minus 20%, in other
words, an amount of around 10% means from 8% to 12%, in particular
a variation of plus or minus 10%, indeed plus or minus 5%.
When the coagulation factor in form of powder, in particular
thrombin, is added, such powder of the coagulation factor is
preferably mixed with the powder of the homogeneous molecular
mixture of collagen/monosaccharide already prepared.
When both a glycosaminoglycan (e.g. chondroitin sulfate) and a
coagulation factor (e.g. thrombin) are added, they are preferably
firstly mixed together, and this mix is added to the previous
mixture of collagen/monosaccharide (already ground into
powder).
The thrombin is not stabilized neither by carbohydrate nor
collagen. The thrombin is never in contact with a solution of the
monosaccharide (contrary to WO 98/57678) which prevents any
denaturation of the protein and a rehydration of the powder leading
to an impossibility to dry it again properly.
The composition in powder form can in particular comprise, or
consist of: particles comprising, or consisting of, collagen of the
fibrillar type and at least one monosaccharide, in particular
glucose, wherein in particular said particles have a size,
granulometry and/or density such as defined in the present
description, and optionally, particles comprising, or consisting
of, at least one glycosaminoglycan, in particular chondroitin
sulfates, and/or at least one coagulation factor, in particular
thrombin, wherein in particular said particles have a size,
granulometry and/or density such as defined in the present
description.
The composition of the hemostatic powder advantageously comprises
at least 50% by weight of particles whose size is between 200 .mu.m
and 400 .mu.m.
The particles constituting the hemostatic powder advantageously
have a mean granulometry ranging from 10 .mu.m to 500 .mu.m, in
particular from 50 .mu.m to 400 .mu.m.
Advantageously, at least 90% by weight, in particular 100% by
weight, of the particles constituting said hemostatic powder can
pass through a screen whose mesh is 500 .mu.m, in particular 400
.mu.m.
At least 90% by weight, and in particular at least 95% by weight,
of the particles constituting said hemostatic powder can be
retained by a screen whose mesh is 10 .mu.m, notably 20 .mu.m,
indeed 30 .mu.m, indeed 50 .mu.m.
This repartition has been chosen to allow the powder to be
hydrated. With particles size too small, the powder does not form a
hydrated matrix consistent with the specification and aspect
required.
The hemostatic composition in powder form comprises in particular:
particles comprising collagen and a monosaccharide, and optionally,
at least glycosaminoglycan and/or a coagulation factor such as
thrombin.
The composition of hemostatic powder can comprise: particles
comprising collagen, a monosaccharide and optionally at least one
glycosaminoglycan and/or coagulation factor, particles comprising
collagen, a monosaccharide and optionally a coagulation factor and
optionally glycosaminoglycan particles, particles comprising
collagen and a monosaccharide and particles comprising at least one
glycosaminoglycan and/or coagulation factor.
In the context of the present invention, the expression "dry
powder" means that the composition comprises a limited content of
solvent, in particular water. Said limited content can be less than
5% by weight, in particular less than 3% by weight, and quite
particularly less than 1% by weight relative to the total weight of
the composition.
Said dry form can be obtained by simple evaporation of the solvent
used, by dehydration by organic solvents.
As indicated above, the described hemostatic powder is formed from
non-cross-linked collagen because it is much simpler in terms of
manufacturing process, and it has been proven to have a good
efficacy with respect to hemostasis even though the collagen of the
powder was not cross-linked.
The inventors have surprisingly discovered that, despite the fact
that the collagen was not cross-linked, mixing the above described
specific hemostatic powder with a saline solution enabled forming
an hemostatic product with a viscosity allowing its direct
application on a bleeding region to promote hemostasis.
This was indeed not expected as it was on the contrary known that
the preparation of an hemostatic collagen paste from a mixture of a
collagen based powder and a saline solution needed the use of
cross-link collagen to work and be stable. This has been in
particular disclosed in the US patent published on Jan. 2, 1990
under the reference U.S. Pat. No. 4,891,359. Cross-linking is
namely known to give stability to the molecules by adding chemical
bonds to the corresponding molecular structure, those additional
chemical bonds being usually required for the molecules to be in an
aqueous form.
Consequently, according to a preferred embodiment, the dry
hemostatic powder as described above, where the collagen is not
cross-linked, is thus to be hydrated with a saline solution in
order to form an hemostatic flowable, which will be applied on the
bleeding region.
The term "flowable" as used herein applies to compositions whose
consistencies enable the composition to sustain a certain shape
without any stress applied, while being deformable if a stress,
such as pressure, is applied on the composition.
A flowable is not a liquid, nor a sponge, nor a powder, rather a
kind of paste, gel or matrix that presents a certain viscosity.
Preferably the flowable has a viscosity comprised between 20 Pas
and 10000 Pas (corresponding to a range of fluidity between 0.0001
(Pas).sup.-1 and 0.05 (Pas).sup.-1).
A flowable refers to a composition that is for instance capable of
passing through a syringe and/or cannula.
In the present description, we refer indifferently to a hemostatic
flowable, a flowable hemostat, and a hemostatic matrix to designate
the same particular composition.
The mixing of the hemostatic powder and the saline solution is thus
performed to make an hemostatic flowable as defined above.
The saline solution is preferably a standard sterile saline
solution used in operating room.
It is preferably composed of distilled water with an amount of
sodium chloride between 0.5% and 1.5%, and preferably around
0.9%.
The saline solution is preferably pure, meaning that it consists of
a mix of sodium chloride in distilled water, without the addition
of any other components.
The saline solution can be stored in different forms, such as bulk
in a large container, or in a specific container of a determined
volume, such as pre-filled syringes.
Preferably, the saline solution is part of a kit to produce the
hemostatic flowable, such a kit also comprising a specific amount
of the hemostatic powder in a container.
Preferably, the hemostatic powder of the kit is stored in a
specific dispenser as illustrated in FIGS. 1 and 2.
Before the hemostatic flowable is prepared, all the active
components are thus contained all together, in a powder form,
within the dispenser. This is very advantageous for several
aspects. It first eases the storage of the product, as one has to
particularly take care of the container having the hemostatic
powder, and not really of the saline solution, which is a commonly
available product. This is also very advantageous in terms of
manufacturing as only the hemostatic powder has to be sterilized
before storage, which would for example not be the case if some
components were first mixed with a saline solution (e.g. thrombin),
and then mixed to an hemostatic powder.
Preferably, the dispenser 1 has a container portion 10 and a nozzle
portion 20 which is adapted for application of the hemostatic
flowable from the container portion 10.
The container portion 10, better illustrated on FIG. 5, has
preferably a shape that favors the mixing of the saline solution
with the hemostatic powder and enhances the hydration speed of the
hemostatic powder.
In addition, the container portion 10 can have bellows 11 that
enables the user to apply the hemostatic flowable by a mere manual
compression of the bellows 11 in order to push the hemostatic
flowable out of the dispenser 1 through the nozzle portion 20.
The nozzle portion 20 is thus designed to allow the hemostatic
flowable to flow out of the dispenser upon compression of the
bellows 11 of the container portion 12.
To ease the manual compression of the bellows 11 by the user, the
nozzle portion 20 can comprise a finger-rest element 21 so that at
least one but preferably two fingers can be positioned on the
finger-rest element 21 to hold the dispenser 1 against the
compression force applied on the bellows 11.
Preferably, as illustrated on FIG. 4, the finger-rest element 21
comprises two elongated members protruding radially from the
longitudinal axis of the dispenser 1, said longitudinal axis
corresponding to the axis of compression of the bellows 11.
The nozzle portion 20 is also preferably designed to enable a user
to fill the container portion 10 with the saline solution through
the nozzle portion 20, which avoids the necessity of removing the
nozzle portion 20 from the container portion 10 and prevents risks
of contamination of the hemostatic powder to be hydrated.
To this end, the nozzle portion 20 may for example comprise a duct
22 through which the saline solution can be injected inside the
container portion 10. Such duct 22 is also designed so that the
hemostatic flowable flows easily out of the dispenser 1, through
the duct 22, when the bellows 11 are compressed.
Preferably, the dispenser further comprises a cap 30 designed to be
removably coupled on the nozzle portion 20.
This cap 30 preferably enables an air-tight seal of the dispenser
1, which is particularly advantageous during the storage and mixing
phases.
When the dispenser 1 is not used the cap 30 is preferably closed as
illustrated on FIG. 1. This can help limit the risks of
contamination of the interior of the device.
The cap 30 used in the dispenser and illustrated on FIG. 3 is
preferably a twist-off cap which eases its removal, opening and
closure.
The amount of hemostatic powder for one kit is preferably between 1
g and 2 g.
The amount of saline solution for one kit is preferably between 4
mL and 10 mL, more preferably between 5 mL and 10 mL.
According to a preferred example, the kit comprises 1.65 g of
hemostatic powder to be mixed with 7 mL of pure saline
solution.
Preferably, in a kit, the mass of saline solution to be used for
hydrating the hemostatic composition is between 2 and 10 times of
the mass of the hemostatic powder, preferably between 4 and 5 times
of the mass of the hemostatic powder.
When a user, e.g. a surgeon, wants to use the proposed hemostatic
flowable, he can prepare it using the kit described above.
To this end, the user opens the application dispenser (also called
applicator) containing the hemostatic powder by removing the
corresponding cap 30.
The user has then to transfer a quantity of sterile saline solution
into the dispenser 1.
Depending on the quantity of hemostatic powder in the dispenser 1,
the quantity of saline solution to be used is between 5 mL and 10
mL, preferably 7 mL.
To transfer the saline solution into the container portion 10 of
the dispenser 1, the user can for instance use a syringe. Such
syringe is preferably provided in the kit, so that it has a volume
corresponding to the exact quantity of saline solution necessary to
form the hemostatic flowable by hydration of the hemostatic
powder.
While transferring the saline solution into the dispenser 1, the
container portion 10 is preferably rotated, for instance around its
own axis, in order to ease the incorporation of the saline solution
into the hemostatic powder. If the saline solution is incorporated
manually by a user, the rotation of the container portion 10 can
also be done by hand. The process could however be automated in
required.
Tapping and/or slightly shaking the container portion 10 while
transferring the saline solution could also be advantageous to
promote incorporation of the saline solution into the hemostatic
powder.
Once the saline solution is transferred into the dispenser 1, the
opening of the nozzle portion 20 is closed, preferably by using the
cap 30 of the dispenser 1, and the container is shaken to mix the
hemostatic powder with the saline solution.
The shaking is preferably done for a duration of at least 15
seconds, even more preferably at least 30 seconds. A shaking time
of between 10 second to 30 seconds, for example 20 seconds, is
however already efficient in terms of hydration of the hemostatic
powder.
The shaking is preferably performed by hand but could also be
automated.
When done manually, the mixing could consist in moving the
dispenser 1 up and down a certain amount of times. For instance,
the dispenser 1 could be moved up and down at least between 10 to
30 times, preferably 20 times. To increase the efficiency of the
mixing, the dispenser 1 could also be flipped over and then moved
up and down a certain amount of times. In this second phase of
manual mixing, the dispenser 1 could also be moved up and down at
least between 10 to 30 times, preferably 20 times.
After the shaking, the dispenser 1 enclosing the hemostatic
flowable having been formed is preferably left to stand for at
least of 30 seconds, preferably at least of 60 seconds, and even
more preferably at least of 90 seconds.
The standing time is likely to be between 30 seconds and 120
seconds, preferably around 90 seconds.
This rest period enables the hydration of the hemostatic powder and
initial swelling to form the hydrated hemostatic flowable.
The hemostatic flowable thus formed has the advantage of being
homogeneous. In particular, the hemostatic flowable has
substantially an homogeneous fluidity within the dispenser. This is
particularly advantageous as the application of the hemostatic
flowable will thus be the same whether it is the beginning of the
product from the dispenser or the remaining of the product.
Once the hemostatic flowable has been formed through hydration of
the hemostatic powder with the saline solution, the hemostatic
flowable is usable for a few hours, e.g. at least 8 hours, without
any loss of properties or performance.
When the hemostatic flowable is ready, it can be used as follows:
Step 1: Blot excess blood from the target bleeding site with a
gauze/pad or suction so that the hemostatic flowable may be applied
directly to the source of bleeding. The wound surface should be as
dry as possible before application. Step 2: Apply the hemostatic
flowable to the source of bleeding by squeezing the bellows. Enough
product should be applied to cover the entire source of bleeding.
Step 3: Immediately use a gauze/pad, preferably wet with saline and
never with blood, to hold the hemostatic material at the target
bleeding site against the bleeding surface, conforming it to the
lesion. Step 4: Maintain the hemostatic material at the target
bleeding site for a certain duration, for example at least two to
three minutes, in order to form a hemostatic clot complex. Gently
lift the gauze and inspect the area. Step 5: If hemostasis has not
been achieved, repeat steps 1-4 or use an alternate method of
hemostasis treatment. Step 6: Discard any unused product after
opening.
Depending on the viscosity (resp. fluidity) of the hemostatic
flowable, step 3 can be avoided, notably if the hemostatic flowable
is viscous enough to hold in place without applying any
gauze/pad.
For better results, it is recommended not to disrupt the clot
complex by physical manipulation.
In addition, once the bleeding has ceased, any excess of the
hemostatic flowable not incorporated in the hemostatic clot should
be removed by gentle irrigation.
One advantage of the hemostatic flowable as proposed is that it can
be used in one time, i.e. by using the whole product contained in
the dispenser at once, or in many times, e.g. when there are
several bleeding regions to treat at the same time or when there
are several sequential bleedings during a surgery.
The hemostatic flowable as described above have the advantage of
enhancing the contact surface with the wound, in particular, the
contact with the bleeding area goes deeper. This is in particular
of interest when the bleeding area corresponds to soft tissues and
parenchymal organs.
In addition, the hemostatic flowable can be easily applied on the
wound or in the bleeding area, for example directly by the surgeon
manual of with a specific applicator. It enables for instance
covering the whole bleeding area without leaving any region
uncovered by the hemostatic flowable.
Another advantage of the proposed hemostatic flowable is that it
fits the habits of the surgeons who are used to treating bleeding
with a hemostatic product in the form of a paste.
This also gives the surgeon the choice to choose the form of the
hemostatic product he wants to use, depending on his habits, on the
specific conditions of surgery, etc. Consequently, he could use
either the hemostatic powder as described in WO 2012/146655, or the
hemostatic flowable as disclosed herein.
The hemostatic powder as described above can be for example
prepared according to a method comprising at least the following
steps: a) formation of an aqueous suspension comprising, preferably
consisting of, collagen of the fibrillar type--mainly comprising
fibrous and/or fibrillar collagen--and a monosaccharide, such as
glucose, b) recovery of the product in the form of precipitate,
paste or gel, notably by centrifugation or decantation, c) drying
of the product, for example by evaporation. d) grinding of the
product to the desired particle-size, in particular by a hammer
mill, and e) optionally, adding thrombin and/or chondroitin
sulfates, notably in solid form, in particular in powder form.
The formation in step a) of an aqueous suspension comprising, the
fibrous/fibrillar collagen and a monosaccharide leads to a
homogeneous repartition of the monosaccharide around the collagen
molecules. Further, the close contact between the molecular species
of collagen and the monosaccharide leads, after dehydration, to a
hard cake suitable for obtaining--by grounding--a powder with the
required high density. On the contrary, mixing a collagen powder
and a glucose powder does not lead to an homogeneous and sprayable
powder, in particular because of the density and electrical
charges.
In step a) the collagen can be present at a concentration ranging
from 30 g/L to 150 g/L.
The monosaccharide can be added to the suspension or to the
homogeneous collagen paste in an amount such as defined in the
description, and more particularly from around 2% to 5% by weight
relative to the weight of the collagen.
In step a) the monosaccharide can be present at a concentration
ranging from 0.3 g/L to 10 g/L.
The aqueous suspension of collagen of step a) can be acid, and in
particular comprise an acid such as hydrochloric acid. Said acid
can be present at a concentration ranging from 0.01 M to 0.5 M, and
in particular from 0.02 M to 0.1 M, indeed around 0.05 M. Said
suspension can be in the form of homogeneous paste.
Step b) can comprise the pouring of the suspension into a mold.
Step c) is performed so as to obtain a cake as thick as possible
(superior the final particle-size wanted), with a very high density
and as less air bubbles as possible (less than 5%) inside the
cake.
Step d) can be followed by a step of screening of the powder,
notably in order to obtain the desired particle size.
According to a preferred embodiment, step a) consists in forming a
mixture comprising 95% by weight of collagen of the fibrillar type
and 5% by weight of glucose. After having dried (step b)) and
ground (step c)) this mixture, chondroitin sulfate is added in a
content of 10% by weight of the total weight of the mixture, such
that the final composition comprises: collagen: 86.36% by weight
relative to the total weight of the composition; glucose: 4.54% by
weight relative to the total weight of the composition; chondroitin
sulfate: 9.09% by weight relative to the total weight of the
composition;
When thrombin is also added, it represents a final content lower
than 0.01% by weight relative to the total weight of the
composition. In the above mixture, thrombin may be in an amount of
0.083 IU/mg of the composition.
For all the aforesaid powder products, it is quite obviously
possible to apply a more or less thorough grinding to obtain a
powder of variable particle-size according to the type of grinding
and the duration thereof.
The hemostatic flowable made from the hemostatic powder mixed with
a specific amount of saline solution can be used as a hemostatic
agent.
This hemostatic flowable can also be used as a pharmaceutical
composition, in particular a hemostatic drug.
As described above, we also propose a hemostatic method comprising
the depositing of the hemostatic flowable such as defined above on
a hemorrhaging part of an animal's body, including humans. In
particular, the hemostatic flowable can be used in surgical
procedures, in particular laparotomies, laparoscopies,
coelioscopies and robotic procedures.
The hemostatic flowable described above could also be used as a
cicatrizing agent for internal and external wounds. The expression
"cicatrizing agent" refers to a product that makes it possible to
obtain a clinically satisfactory cicatrization of the tissues with
which it is in contact.
EXAMPLES
Example 1: Protocol for Measuring Hemostatic Capacity In Vitro
Citrated (around 0.1 M) human blood is maintained at 37.degree. C.
in a water bath throughout the measurement. The product to be
tested (10 mg) is deposited in a 5 mL polypropylene tube with a
snap-on cap, and then citrated fresh blood (2 mL) is added.
CaCl.sub.2) is then added so that the final CaCl.sub.2)
concentration in the blood is 15 mM, and then the test tube is
closed. The contents are then around mixed by vigorous inversions
(10 times) and then the test tube is plunged into the water bath;
the test tube is returned to the vertical position every 10
seconds. The time required to form a clot is noted and corresponds
to hemostatic capacity.
Example 2: Protocol for Measuring Particle Size
A known quantity of product, notably of powder, is sifted through
50 .mu.m, 100 .mu.m, 200 .mu.m, 300 .mu.m and 400 .mu.m screens for
2 minutes (per screen). The fractions from each screen are weighed.
The proportion of each particle size range is determined.
Example 3: Protocol for Measuring the Swelling of the
Composition
A 15 mL flask is weighed (m.sub.0 in mg) and then X mg of powder of
the dry composition is added (m.sub.0+X in mg). A 0.15 M aqueous
NaCl solution (2 mL) is added and the composition is left to swell
for 20 minutes; the flask is then centrifuged at 1,000 rpm.
Excess NaCl is removed with a Pasteur pipette and droplets are
eliminated by turning over the flask on filter paper; the flask is
then weighed with the wet powder (m.sub.1 in mg).
The swelling ratio is calculated as follows:
((m.sub.1-m.sub.0)/(m.sub.0+X-m.sub.0)).
Example 4: Preparation of Collagen of the Fibrillar Type by Basic
Extraction
Pieces of pig dermis (30 kg), defatted with acetone, are left to
swell for 3 hours in 100 kg of 0.05 M NaOH solution. The dermises
are finely cut up by a cutting mill and the paste obtained is
diluted with 50 liters of 0.05 M NaOH. The mixture is then sieved
under pressure through a 1 mm screen. The paste obtained is then
brought to pH 6-7.5 with HCl and the precipitate obtained is
collected by centrifugation or filtration through a 1 mm
screen.
The retentate is dehydrated with acetone according to methods known
to those persons skilled in the art. This dehydrated retentate thus
consists in collagen of the fibrillar type, with a large content of
fibrillar/fibrous collagen relative to the non-fibrillar collagen.
Generally, such extracted collagen comprises from 85% to 95% by
weight of fibrillar/fibrous collagen relative to the total weight
of the collagen, and from 5% to 15% by weight of non-fibrillar
collagen relative to the total weight of the collagen.
Example 5: Preparation of a Hemostatic Powder #1
30 g of collagen of the fibrillar type as prepared in Example 4 is
added to 1 L of a 0.02 M aqueous HCl solution and the mixture is
then stirred for 5 hours. Next, to the homogeneous paste obtained,
powdered fructose is added in an amount of 2% (0.6 g) by weight
relative to the weight of the collagen.
The mixture is homogenized for 1 hour and then poured out and
dehydrated. After drying, the dry product is ground at a rate of 25
g/min using a Fitzpatrick hammer mill at 7,000 rpm under controlled
heating. The product is then screened by mechanical sifting to
eliminate particles whose size is larger than 400 .mu.m.
Dermatan sulfate is then added to the powder in an amount of 2% by
weight relative to the dry matter of the powder (0.612 g).
The mixture is then homogenized using a ball mill, lyophilized
thrombin is added to the mixture in an amount of 15 IU/mg of
powder, and finally the mixture is homogenized using a ball
mill.
Example 6: Preparation of a Hemostatic Powder #2
7.5 kg of collagen of the fibrillar type as prepared in Example 4
is added to 50 L of a 0.05 M aqueous HCl solution and the mixture
is then stirred for 16 hours. Next, to the homogeneous paste
obtained, powdered fructose is added in an amount of 5% (375 g) by
weight relative to the weight of the collagen.
The mixture is homogenized for 3 hours and then distributed onto
plates and dehydrated. After drying, the dry product is ground by
fraction at a rate of 5 g/min using a hammer mill at 12,000 rpm
under controlled heating. The product is then screened by
mechanical sifting to eliminate particles whose size is larger than
400 .mu.m and those smaller than 50 .mu.m.
Granulometry is measured in order to verify that the distribution
is such that 60% of the sample by weight has a granulometry greater
than 200 .mu.m.
Purified chondroitin sulfates are then added to the powder in an
amount of 20% by weight relative to the dry matter of the powder
(1.575 kg). The mixture is homogenized using a ball mill.
Finally, lyophilized thrombin is added to the mixture in an amount
of 10 IU/mg of powder. As before, the mixture is homogenized using
a ball mill.
Example 7: Preparation of a Hemostatic Powder #3
1000 g of collagen of the fibrillar type as prepared in Example 4
is added to 60 mL of a 0.02 M aqueous HCl solution and the mixture
is then stirred for 5 hours. Next, to the homogeneous paste
obtained, powdered glucose is added in an amount of 5% (50 g) by
weight relative to the weight of the collagen.
The mixture is homogenized for 1 hour and then poured out and
dehydrated. After drying, the dry product is ground at a rate of 25
g/min using a Fitzpatrick hammer mill at 7,000 rpm under controlled
heating. The product is then screened by mechanical sifting to
eliminate particles whose size is larger than 400 .mu.m and smaller
than 50 .mu.m.
Chondroitin sulfate is then added to the powder in an amount of 10%
by weight relative to the dry matter of the powder (105 g). The
mixture is then homogenized using a ball mill.
Such powder composition has a tapped density of around 0.408
g/mL.
Example 8: Preparation of a Hemostatic Powder #4
500 g of collagen of the fibrillar type as prepared in Example 4 is
added to 30 mL of a 0.02 M aqueous HCl solution and the mixture is
then stirred for 5 hours. Next, to the homogeneous paste obtained,
powdered glucose is added in an amount of 5% (25 g) by weight
relative to the weight of the collagen.
The mixture is homogenized for 1 hour and then poured out and
dehydrated. After drying, the dry product is ground at a rate of 25
g/min using a Fitzpatrick hammer mill at 7,000 rpm under controlled
heating. The product is then screened by mechanical sifting to
eliminate particles whose size is larger than 400 .mu.m and smaller
than 50 .mu.m.
Chondroitin sulfate mixed with a thrombin powder is then added to
the powder in an amount of 10% by weight relative to the dry matter
of the powder (52.5 g). The thrombin is added to the mixture in a
final amount of 0.85 U/mg. The mixture is then homogenized using a
ball mill.
Such powder composition has a tapped density of around 0.425
g/mL.
Example 9: Preparation of a Hemostatic Powder #5
750 g of collagen of the fibrillar type as prepared in Example 4 is
mixed with 6675 mL of highly purified water. The mixture is stirred
at a first stirring rate of 20 rpm during 10 minutes, and then at a
second stirring rate of 40 rpm during 15 minutes.
The above mixture is then stirred again at the first stirring rate
of 20 rpm while a solution of glucose (37.5 g of glucose with 300
mL of water) is incorporated. The quantity of glucose added
corresponds to 5% by weight relative to the weight of the collagen
being used in the mixture. This new mixture is stirred at the
second stirring rate of 40 rpm during 10 minutes. This preparation
is then stored during 16 hours.
A quantity of 87.5 mL of a 1 M aqueous HCl solution is then added
to the preparation while being stirred at a stirring rate of 30
rpm. This new mixture is then stirred at a first stirring rate of
35 rpm during 1 minute, then at a second stirring rate of 40 rpm
during 1 minute, followed then by several stirring sessions of 5
minutes at the same stirring rate of 40 rpm, a quick pause in the
stirring being made between two sessions.
The thick paste obtained in the preceding phase is then separated
in several pieces having similar shape and mass. Those pieces of
the paste are then placed for 24 hours in a hermetically sealed
enclosure having an atmosphere saturated with ammonia. After this
neutralization step, the pieces of paste are dried at 20.degree. C.
during 96 hours, and the dry products are then ground at a rate of
1 kg/h using a cryogenic mill of Forplex at 8,500 rpm. The powder
product is then screened by mechanical sifting to eliminate
particles whose size is larger than 200 .mu.m and smaller than 50
.mu.m, resulting in a collagen-glucose powder.
A powder of chondroitin sulfate (CS) which is made of particles
having a size between 50 .mu.m and 200 .mu.m is then added to the
collagen-glucose powder in an amount of 10% by weight relative to
the dry matter of the collagen-glucose powder. For instance, 30 g
of the powder of chondroitin sulfate is mixed with 300 g of the
collagen-glucose powder. For this hemostatic powder #5,
freeze-dried thrombin is also added, in a quantity of 1000 UI/g.
The mixture is then homogenized using a V blender. The final
hemostatic powder has a tapped density of around 0.4 g/mL.
Example 10: Collagen Characterization.fwdarw.Presence of Soluble
Collagen in the Collagen, Determination of the Ratio Between
Fibrillar/Fibrous Collagen and Non-Fibrillar Collagen
The goal of the experimentation is to determine the proportion of
fibrillar/fibrous collagen and non-fibrillar collagen in a collagen
(extracted collagen or collagen ground into powder). Such
proportion can be determined by studying the proportion of
insoluble (corresponding to the fibrillar/fibrous collagen) and
soluble collagen (corresponding to the non-fibrillar collagen) in
the collagen.
The experimentation consists in solubilizing about 2.5 g of the
collagen under test in 166 mL of water at pH 13 during 16 hours.
The solution is then centrifuged (10 000 rpm during 10 minutes).
The supernatant (corresponding to the non-fibrillar collagen) and
the residue (corresponding to the fibrous/fibrillar collagen) are
then split. The residue is directly dried with successive acetone
baths and under a controlled air flow. The pH of the supernatant is
adjusted at pH 3 with acetic acid and chlorhydric acid at 6M. The
solid collagen from the supernatant is obtained by adding NaCl
0.6M, and by performing a centrifugation. It is then dried with
successive acetone baths and under a controlled airflow.
The collagen weights from the residue (Mresidue) and from the
supernatant (Msupernatant) are calculated, and the formula
Mresidue/(Mresidues+Msupernatant).times.100 gives the percentage of
fibrous collagen on total amount of collagen.
In the invention, the ratio Mresidue/(MResidues+Msupernatant) must
be superior to 80% both for the collagen used to prepare the powder
and for the final collagen powder. Preferentially the ratio is
superior to 85%.
For example, the above experimentation made of three batches of
collagen prepared as in example 4 gives very similar ratios of
92.67%, 94.60% and 91.51% respectively. After having ground the
collagen of these three batches, the ratio remains very similar as
it is of 91.63%, 88.02%, and 88.69% respectively.
Another way to show the presence of both fibrous/fibrillar collagen
and soluble collagen is to perform a SDS page electrophoresis.
FIG. 6 illustrates such electrophoresis, with sample S1
corresponding to the supernatant of a first batch (made from
collagen extracted as in example 4), sample S2 corresponding to the
residue of this first batch, and sample S3 corresponding to the
supernatant of a second batch (also made from collagen extracted as
in example 4), sample S4 corresponding to the residue of this
second batch.
The results show that for the collagen from the residue, a larger
amount of fiber cannot migrate through the acrylamide gel and are
stained at the stop of the gel. The preparation of the sample does
not allow the split of each chain from the collagen. Therefore,
alpha chains are present in a very low amount. The collagen from
the supernatant is able to entirely migrate in the gel, there are
no fiber blocked at the top, chains from the collagen are properly
split during the electrophoresis process.
BIBLIOGRAPHIC DATA
WO 2012/146655 "Nature designs tough collagen: explaining the
nanostructure of collagen fibrils," by Markus Buehler (PNAS, Aug.
15, 2006, vol. 103, no. 33, pp. 12285-12290) "Extraction of
collagen from connective tissue by neutral salt solutions" by
Jerome Gross, John H. Highberger and Francis O. Schmitt
(Proceedings of the NATIONAL ACADEMY OF SCIENCES Volume 41 Number I
Jan. 15, 1955) WO 2010/125086 FR2944706 WO 01/97873 U.S. Pat. No.
4,891,359
* * * * *
References